The skin is made of two layers, dermis and epidermis. The epidermis is the outermost layer and is composed of keratinocytes, melanocytes and Langerhans cells. The main cell population in the epidermis consists of keratinocytes, which form a keratinized layer that is constantly renewed. Its function is to protect against external agents, whether physical, chemical or pathogenic. The dermis is located more internally and is attached to the epidermis through the basement membrane. It consists of fibroblasts, adipocytes and macrophages, is irrigated by blood vessels and has many nerve endings responsible for transmitting sensations of touch and temperature. The hair follicles lay in the dermis, as well as the sweat, sebaceous and apocrine glands, whose function is to maintain the integrity and elasticity of the skin. These properties are given by their extracellular matrix, composed of proteins secreted by the fibroblasts.
The proteins of the extracellular matrix (ECM) are classified into two groups: glycosaminoglycans and fibrous proteins. Glycosaminoglycans (GAG) are unbranched chains resulting from the polymerization of aminosugar disaccharides. Due to their chemical properties and their large number of negative charges, GAG form bulky structures which tend to attract large amounts of water, giving the ECM resistance to compression. Fibrous proteins have structural and adhesive functions and are mainly two: elastin and collagen, which are responsible for the mechanical properties of the tissues such as the ability to resist tension, compression, extensibility and torsion.
The elasticity and resilience properties of ECM are due to a network of elastic fibers. Structurally, the elastic fibers are composed of an elastin core covered by a pod of microfibrils of approximately 10 nm in diameter. Microfibrils are composed of fibrillin and microfibril-associated glycoprotein (MAGP). The assembly of the elastic fibers is sequential, microfibrils appearing first and forming a skeleton on which elastin is deposited. Elastin is a highly hydrophobic protein, composed of approximately 750 amino acid residues and is originated from a hydrosoluble precursor, tropoelastin, which is secreted into the extracellular space by the fibroblasts. Elastin fibers are the result of the assembly and crosslinking of tropoelastin monomers near the plasma membrane of the fibroblasts.
The tropoelastin molecule is synthesized in soluble form, with a molecular weight of about 70 kDa, and in its sequence presents hydrophobic domains alternating with crosslinking domains [Brown-Augsburger P., Tisdale C., Broekelmann T., Sloan C. and Mecham R. P. (1995) “Identification of an elastin crosslinking domain that joins three peptide chains” J. Biol. Chem. 270:17778-17783]. The hydrophobic domains are repetitions of peptides from two to nine amino acids rich in proline, alanine, valine, leucine, isoleucine and glycine, among which valine and glycine are especially abundant [Debelle L. and Tamburro A. M. (1999) “Elastin: molecular description and function” Int J. Biochem. Cell Biol. 31:261-272]. The interactions between hydrophobic domains are important in the assembly and essential for the elasticity of the molecule [Bellingham C. M., Woodhouse K. A., Robson P., Rothstein S. J. and Keeley F. W. (2001) “Self-aggregation of recombinantly expressed human elastin polypeptides” Biochim. Biophys. Acta. 1550:6-19]. The crosslinking domains of tropoelastin contain lysine residues within proline-rich regions or polyalanine regions. The formation of covalent crosslinks of desmosine by lysyl oxidase action stabilizes the polymerized and insoluble product [Csiszar K. (2001) “Lysyl oxidases: a novel multifunctional amine oxidase family” Prog. Nucleic Acid Res. Mol. Biol. 70:1-32] and only two oxidase lysyl proteins, called LOX and LOXL, are capable of crosslinking insoluble elastin [Borel A., Eichenberger D., Farjanel J., Kessler. E., Gleyzal C., Hulmes D. J. S., Sommer P. and Font B. (2001) “Lysyl oxidase-like protein from bovine aorta” J. Biol. Chem. 276:48944-48949]. Additionally, the translated sequence of tropoelastin has a negatively charged hydrophilic C-terminal domain which is highly conserved between species. The major post-translational modifications that affect this molecule are hydroxylations of proline residues.
Elastogenesis is the process which leads to the generation of functional elastin in elastic fibers. It begins within the cell with the synthesis of the tropoelastin molecule, to which a galactose lectin of 67 kDa is added, which acts as a chaperone preventing the intracellular aggregation of tropoelastin molecules. The complex is secreted into the extracellular space, where galactose lectin interacts with the galactosugar of the microfibrils, thereby reducing its affinity for tropoelastin, which is locally released. The galactose lectin of 67 kDa is recycled and may resume its function, whereas tropoelastin is deposited in the skeleton formed by microfibrillar components through the interaction of the N-terminal domain of microfibril-associated glycoprotein (MAGP) with the C-terminal domain of tropoelastin. Once aligned, the majority of lysine residues of tropoelastin are deaminated and oxidized to aldehydes by the action of the Cu2+-dependent lysyl oxidase. Crosslinking occurs throughout the reaction of said formed aldehydes with themselves or with unmodified lysine, and consequently tropoelastin chains become insoluble and the elastin network grows. Molecules belonging to the emiline and fibulin families, which are believed to possibly modulate the deposition of tropoelastin on microfibrils, are found in cell membrane-elastic fiber and elastin-microfibril interfaces. There is presently no evidence that molecules other than fibrillin are indispensable for the assembly of microfibrils [Kielty C. M., Sherratt M. J. and Shuttleworth C. A. (2002) “Elastic fibers” J. Cell. Sci. 115:2817-2828]. Mature elastin is an insoluble polymer of tropoelastins covalently joined by crosslinks, which may be bi-, tri- or tetrafunctional. It is believed that the complexity increases over time. The hydrophobic fragments are highly mobile and heavily contribute to the entropy of the system, and the amount of water hydrating the polymer in vivo also contributes to the entropy of the system [Debelle L. and Tamburro A. M. (1999) “Elastin: molecular description and function” Int. J. Biochem. Cell. Biol. 31:261-272].
Elastic fibers are important for maintaining skin elasticity, but also in other tissues and organs, like lungs or large blood vessel walls [Faury G. (2001) “Function-structure relationship of elastic arteries in evolution: from microfibrils to elastin and elastic fibers” Pathol. Biol. (Paris) 49:310-325]. Defects in the formation of elastic fibers, such as mutations in the genes that encode the different proteins which compose them, result in different pathologies. Thus, mutations in the fibrillin-1 gene are responsible for the Marfan syndrome (associated with skeletal, ocular and cardiovascular symptoms); mutations in the fibrillin-2 gene lead to congenital contractural arachnodactyly in addition to ocular and skeletal symptoms, and mutations in the elastin gene are the cause of the Williams syndrome, supravalvular stenosis and cutis laxa [Tassabehji M., Metcalfe K., Hurst J., Ashcroft G. S., Kielty C., Wilmot C., Donnai D., Read A. P. and Jones C. J. (1998) “An elastin gene mutation producing abnormal tropoelastin and abnormal elastic fibers in a patient with autosomal dominant cutis laxa” Hum. Mol. Genet. 6:1021-1028].
Elastic fibers have the purpose of maintaining flexibility throughout the life of a person. However, there are enzymes that may degrade them, resulting in loss of skin elasticity, which is a factor that significantly contributes to the aging of connective tissues and plays an important role in the degeneration of the skin by sun exposure [Watson R. E. B. Griffiths C. E. M., Craven N. M., Shuttleworth C. A. and Kielty C. M. (1999) “Fibrillin-rich microfibrils are reduced in photoaged skin. Distribution at the dermal-epidermal junction” J. Invest. Dermatol. 112:782-787].
Elastase is an enzyme that belongs to the family of serine proteases, which has carbohydrates attached to its surface. Its biological function is the degradation of elastin to allow neutrophil migration through connective tissues, so that they can destroy pathogenic microorganisms in case of infection. In humans, there are two genes that encode elastase, the pancreatic elastase gene and the neutrophil elastase gene, and elastase is produced and secreted by cells such as neutrophils, macrophages, fibroblasts and pancreatic cells. Other forms of elastase are found in microorganisms and in the venom of some snakes. The action of elastase may involve a decrease in the elasticity, health and quality of the skin. Several disorders have been described in which elastase activity is the direct or indirect cause of skin symptoms associated to it, such as wrinkles and stretch marks due to aging and photoaging, bullous pemphigoid, dermatitis and psoriasis. Elastase activity is also related with wound healing disorders such as keloids and hypertrophic scars, and skin alterations resulting from its lack of elasticity, like orange peel skin in cases of cellulite. Elastic fibers, due to their low rate of remodeling, appear at sites of trauma after some time, i.e. are absent in recent scars and their arrangement is abnormal in mature scars.
Collagens are the main family of fibrous proteins of the extracellular matrix, constituting 25% of the total proteic mass in mammals. They have been classified in more than 20 families, all of them having individual characteristics which fulfill specific functions in different tissues.
The main characteristic of collagen is its helicoidal structure formed by the association of three polypeptide chains rich in glycine and proline. Alterations in its amino acid composition cause dysfunction and loss of its mechanical properties [Culav E. M., Clark C. H. and Merrilees M. J. (1999) “Connective tissues: matrix composition and its relevance to physical therapy” Phys. Ther. 79:308-319]. These polypeptide chains can associate one to the other and form fibrils, which have a diameter of 10-300 nm and a length of up to hundreds of micrometers in mature tissues. These fibrils are often added into major structures, such as cable bunching, which can be seen through electronic microscopy as collagen fibers of several micrometers in diameter. This process is known as fibrillogenesis [Aumailley M. and Gayraud B. (1998) “Structure and biological activity of the extracellular matrix” J. Mol. Med. 76:253-265]. Not all collagens have the ability to form fibrils; only I, II, III, V and XI type collagens, which are known as fibrillar collagens.
An adult dermis is basically formed by fibrillar collagens type I, III and V. Type I collagen represents 80-90% of the total collagen of the dermis. Generally, type I collagen fibers feature a bigger diameter, which correlates with its ability to withstand a bigger mechanical load. Type III collagen intervenes in tissue extensibility, and with aging, it is replaced by type I collagen molecules, a process which is partly responsible for mature skins being less extensible than young skins. Type V collagen associates with types I and III regulating the diameter of fibrils [“The Biology of the Skin”, Freinkel R. K. and Woodley D. T., eds. The Parthenon Publishing Group, 2001; Culav E. M., Clark C. H. and Merrilees M. J. (1999) “Connective tissues: matrix composition and its relevance to physical therapy” Phys. Ther. 79:308-319].
Collagen fibers are in constant renewal process, but such renewal decreases with age, causing the thinning of dermis. In addition, even though collagen fibers organization provides collagen network with great resistance, collagen fibers are sensitive to certain enzymes known as matrix metalloproteases (MMP). MMPs belong to a family of proteolytic enzymes (endoproteases) which contain a zinc atom coordinated with three cysteine residues and one residue of methionine in its active center and which can, collectively, degrade macromolecular components from the extracellular matrix and from the basal laminas into a neutral pH (collagen, elastin, etc.).
The solidity of the dermis is mainly due to the overlapping of the collagen fibers packed against each other in all directions. The collagen fibers contribute towards the elasticity and tonicity of the skin and/or mucous membranes. However, the thinning of the dermis is due not only to chronological aging but also to pathological causes such as, for example, the hypersecretion of corticoid hormones, certain diseases (Marfan's syndrome, Ehlers-Danlos syndrome) or vitamin deficiencies (scurvy). It is also accepted that extrinsic factors such as ultraviolet radiation, tobacco or certain treatments such as retinoic acid and derivatives, glucocorticoids or vitamin D and derivatives, also have an effect on the skin, mucous membranes and/or scalp and on its level of collagen. Degradation of the collagen fibers results in the appearance of loose, wrinkled skin, particularly on those skin areas exposed to the solar light such as the face, ears, neck, scalp, arms and hands, which people have always tried to combat, since skin which looks smooth and taut is preferred.
Skin damage associated to chronic exposition (repetitive irradiation) or high exposition (strong irradiation) to UVA and/or UVB rays has been studied; particularly it is known that UVB rays (290-300 nm; 5% of total UV rays) with more energetic wavelength especially affect epidermic cells (keratinocytes) acting over its DNA, and that UVA rays (320-400 nm; 95% of total UV rays) have a stronger penetration grade and also act over dermic cells such as fibroblasts and they act indirectly generating free radicals.
Moreover, prolonged exposure to UV radiation, particularly to UVA and/or UVB radiation stimulates MMP expression which destroy collagen [Fisher G. J., Datta S. C., Talwar H. S., Wang Z. Q., Varani J., Kang S, and Voorhees J. J. (1996) “Molecular basis of sun-induced premature skin ageing and retinoid antagonism” Nature 379:335-339; Fisher G. J., Wang Z. Q., Datta S. C., Varani J., Kang S, and Voorhees J. J. (1997) “Pathophysiology of Prematur Skin Aging Induced by Ultraviolet Light” New Eng. J. Med. 337:14191429; Fisher G. J., Choi H. C., Bata-Csorgo Z., Shao Y., Datta S., Wang Z. Q., Kang S, and Voorhees J. J. (2001) “Ultraviolet irradiation increases matrix metalloproteinase-8 protein in human skin in vivo” J. Invest. Dermatol. 117:219-226], especially matrix metalloelastase type 1 (MMP-1). This is one of the components of photoinduced skin aging or photoaging [Rittie L. and Fisher G. J. (2002) “UV-light-induced signal cascades and skin aging” Ageing Res. Rev. 1:705-720]. Besides, it is known that MMP-1, MMP-2 and MMP-9 activity increases with age and that this increase, together with cell growth deceleration, contributes to chronologic skin aging [EP 1 005 333 81]. Similarly, smokers' skin also has a premature aging aspect in which MMPs are overexpressed [Lahmann C., Bergemann J., Harrison G. and Young A. R. (2001) “Matrix metalloproteinase-1 and skin aging in smokers” Lancet 357:935-936].
Elastic fibers increase their crosslinks with time, decreasing the general elasticity and in parallel the progressive fragmentation of elastin causes a reduction of dermis density. These changes involve a loss of skin elasticity and the appearance of the hallmarks of aging. Moreover, the damage of the extracellular matrix induced by UV light leads to the appearance of wrinkles, loss of skin resilience and to actinic elastosis. Elastosis macroscopically appears as frequent yellowish nodules on the exposed skin. At the histological level, elastosis means an accumulation of basophilic amorphous material in the papillary dermis composed of elastin, microfibril proteins, fibronectin and type I and III collagen.
The clearest sign of aging is the appearance of wrinkles, which partially is due to the loss of skin elasticity. At the molecular level, elastic fibers have a linear appearance in young skin, which is indicative of elasticity. Crooked or curly fibers imply a lack of elasticity and are associated with age [Imokawa G., Takema Y, Yorimoto Y., Tsukahara K., Kawai M. and Imayama S. (1995) “Degree of UV-induced tortuosity of elastic fibers in rat skin is age dependent” J. Invest. Dermatol. 105:254-258]. The repair of skin wrinkling induced by agents such as trans-retinoic acid or CO2 laser involves a recovery of the linearity of the elastic fibers [Tsukahara K., Takema Y., Moriwaki S., Fujimura T., Imayama S, and Imokawa G. (2001) “Carbon dioxide laser treatment promotes repair of the three-dimensional network of elastic fibers in rat skin” Br. J. Dermatol. 144:452-458, Tsukahara K., Takema Y, Fujimura T., Moriwaki S., Kitahara T., Imayama S, and Imokawa G. (1999) “All-trans retinoic acid promotes the repair of tortuosity of elastic fibers in rat skin” Br. J. Dermatol. 140:1048-1053]. The loss of linearity of elastic fibers may be due to secretion of elastase by surrounding fibroblasts, and may be accompanied by the absence of endogenous elastase inhibitors. Moreover, ultraviolet B (UVB) radiation can cause that the fibroblasts stop maintaining tension on the elastic fibers, making them lose linearity, with the consequent loss of flexibility. It has been shown that specific inhibition of skin elastase inhibits wrinkles, delays the loss of skin elasticity and slows the degradation of the three-dimensional structure of elastic fibers [Tsukahara K., Takema Y, Moriwaki S., Tsuji N., Suzuki Y., Fujimura T. and Imokawa G. (2001) “Selective inhibition of skin fibroblast elastase elicits a concentration-dependent prevention of ultraviolet B-induced wrinkle formation” J. Invest. Dermatol. 117:671-677]. The inhibition of elastase activity by topical application, therefore, can reduce, prevent or delay symptoms of aging and photoaging such as wrinkles, marks and expression lines.
Moreover, during the menopause, the main changes relating to the dermis are a decrease in the level of collagen and in the thickness of the dermis. In menopausal women, this results in thinning of the skin and/or mucous membranes. Women thus experience a sensation of “dry skin” or of skin which feels tight and an increase in the level of surface wrinkles and fine lines is observed. The skin looks rough to the touch. Lastly, the skin is less supple. It is demonstrated that women lose 2.1% of their level of collagen per year after the menopause and that 30% is lost in the first five years after the menopause [Brincat M., Kabalan S., Studd J. W., Moniz C. F., de Trafford J. and Montgomery J. (1987) “A study of the decrease of skin collagen content, skin thickness, and bone mass in the postmenopausal woman” Obstet. Gynecol. 70:840-845].
Proteases play an important role in different skin, mucous membranes and/or scalp conditions and disorders in which there is a degradation and destruction of collagen and/or elastin [Kahari V. M. and Saarialho-Kere U. (1997) “Matrix metalloproteinases in skin” Exp. Dermatol. 6:199-213]. Among the different pathologies described in which there is a degradation of collagen due to the activity of proteases in connective tissue cells, we find chronic ulcer [Miyoshi H., Kanekura T., Aoki T. and Kanzaki T. (2005) “Beneficial effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on chronic dermatitis” J. Dermatol. 32:346-353], psoriasis [Flisiak I., Mysliwiec H. and Chodynicka B. (2005) “Effect of psoriasis treatment on plasma concentrations of metalloproteinase-1 and tissue inhibitor of metalloproteinase-1” J. Eur. Acad. Dermatol. Venereol. 9:418-421; Suomela S., Kariniemi A. L., Impola U., Karvonen S. L., Snellman E., Uurasmaa T., Peltonen J., Saarialho-Kere U. (2003) “Matrix metalloproteinase-19 is expressed by keratinocytes in psoriasis” Acta Derm. Venereol. 83:108-114], oral pathologies such as gingivitis and periodontitis [Reynolds J. J. and Meikle M. C. (1997) “The functional balance of metalloproteinases and inhibitors in tissue degradation: relevance to oral pathologies” J. R. Coll. Surg. Edinb. 42:154-160], skin cancer [Ntayi C., Hornebeck W. and Bernard P. (2004) “Involvement of matrix metalloproteinases (MMPs) in cutaneous melanoma progression” Pathol. Biol. (Paris) 52:154-159; Kerkela E. and Saarialho-Kere U. (2003) “Matrix metalloproteinases in tumor progression: focus on basal and squamous cell skin cancer” Exp. Dermatol. 12:109-125] and tumor invasion and metastasis [Sato H., Takino T. and Miyamori H. (2005) “Roles of membrane-type matrix metalloproteinase-1 in tumor invasion and metastasis” Cancer Sci. 96:212-217].
Proteases also play a key role in different physiological situations in which the extracellular matrix is degraded or reconstructed, such as the extracellular matrix proteolytic remodeling, including tissue morphogenesis during development, tissue repair and angiogenesis [Kahari V. M. and Saarialho-Kere U. (1997) “Matrix metalloproteinases in skin” Exp. Dermatol. 6:199-213]. In a particular way, MMPs have a crucial role in connective tissue remodeling [Abraham D., Ponticos M. and Nagase H. (2005) “Connective tissue remodeling: cross-talk between endothelins and matrix metalloproteinases” Curr. Vasc. Pharmacol. 3:369-379], for example collagen degradation by MMPs makes the skin look wrinkled and flaccid.
Another skin and/or scalp pathologies or disorders, associated to MMP overexpression or to an increase of MMP activity in the connective tissue is acne [Papakonstantinou E., Aletras A. J., Glass E., Tsogas P., Dionyssopoulos A., Adjaye J., Fimmel S., Gouvousis P., Herwig R., Lehrach H., Zouboulis C. C. and Karakiulakis G. (2005) “Matrix metalloproteinases of epithelial origin in facial sebum of patients with acne and their regulation by isotretinoin” J. Invest. Dermatol. 125:673-684]. It is described that skins affected by acne have high levels of MMP-1.
Fibroblasts are not the only type of cells secreting elastase, elastase produced by neutrophils also contributes to the appearance of skin disorders or even pathologies. Neutrophil elastase or HLE (Human Leukocyte Elastase) is a proinflammatory agent that is known to be capable of degrading various components of extracellular matrix such as elastin, type III and IV collagen, and proteoglycans. The activity of neutrophil elastase is increased in the surface of the diseased skin of patients with psoriasis, atopic dermatitis and allergic contact dermatitis [Wiedow O., Wiese F., Streit V., Kalm C. and Christophers E. (1992) “Lesional elastase activity in psoriasis, contact dermatitis, and atopic dermatitis,” J. Invest. Dermatol. 99:306-309], pathologies characterized by leukocyte infiltration of the skin. Leukocyte infiltration of the skin, moreover, is increased by UV light [Woodbury R. A., Kligman L. H., Woodbury M. J. and Kligman A. M. (1994) “Rapid assay of the anti-inflammatory activity of topical corticosteroids by inhibition of a UVA-induced neutrophil infiltration in hairless mouse skin. I. The assay and its sensitivity “Acta Derm. Venereol. 74:15-17]. Furthermore, elastin degradation by elastase generates elastin fragments that act as cytokines contributing to a chronic inflammatory state associated with aging [Antonicelli F., Bellon G., Debelle L. and Hornebeck W (2007) “Elastin-elastase and inflamm-aging” Curr. Top. Dev. Biol. 79:99-155].
Psoriasis is a chronic skin inflammation whose cause is unknown. Its appearance is related to an inflammatory response mediated by elements of the immune system. The most characteristic feature of this pathology is the appearance of hyperplastic keratinized lesions accompanied by lymphocytic infiltration. These lesions expand at the edges, where there is a greater proliferative activity. It has been shown that elastase induces keratinocyte proliferation [Rogalski C., Meyer-Hoffert U., Proksch E. and Wiedow O. (2002) “Human leukocyte elastase induces keratinocyte proliferation in vitro and in vivo” J. Invest. Dermatol. 118:49-54]. These data indicate that dermal formulations containing compounds that inhibit the elastase enzyme activity are an adequate approximation to alleviate the symptoms of affected skin that occur with inflammation and leukocyte infiltration together with increased levels of elastase, for example and not limited to, for the treatment of psoriasis, atopic dermatitis and allergic contact dermatitis. People with dermatitis, including contact dermatitis and atopic dermatitis, also have high levels of some MMPs [Herouy Y., Mellios P., Bandemir E., Dichmann S., Nockowski P., Schöpf E. and Norgauer J. (2001) “Inflammation in stasis dermatitis upregulates MMP-1, MMP-2 and MMP-13 expression” J. Dermatol. Sci. 25:198-205; Devillers A. C., van Toorenenbergen A. W., Klein Heerenbrink G. J., Muldert P. G. and Oranje A. P. (2007) “Elevated levels of plasma matrix metalloproteinase-9 in patients with atopic dermatitis: a pilot study” Clin. Exp. Dermatol. 32:311-313; Miyoshi H., Kanekura T., Aoki T. and Kanzaki T. (2005) “Beneficial effects of tissue inhibitor of metalloproteinases-2 (TIMP-2) on chronic dermatitis” J. Dermatol. 32:346-353]. “Dermatitis” is defined as those skin conditions, disorders or pathologies that cause inflammation, including contact dermatitis, atopic dermatitis, sensitive skin and eczema.
Likewise, rosacea is a skin and/or scalp pathology or disorder in which MMPs are also involved. Rosacea is characterized by an increase of angiogenesis and inflammation. Angiogenesis refers to the process of new blood vessels formation and it includes benign conditions such as rosacea and malignant processes such as cancer. Matrix degrading enzymes, present in tissue extracellular matrix facilitate angiogenesis since they allow new blood vessels to penetrate the matrix. MMPs represent a kind of enzymes involved in such processes [Sapadin A. N., Fleischmajer R. (2006) “Tetracyclines: Nonantibiotic properties and their clinical implications” J. Am. Acad. Derm. 54:258-265].
Another inflammatory skin disease is bullous pemphigoid, which causes blistering. It has been demonstrated that neutrophils are involved in the onset of skin lesions in bullous pemphigoid, and their action is mediated by elastase [Liu Z., Shapiro S. D., Zhou X., Twining S. S., Senior R. M., Giudice G. J., Fairley J. A. and Diaz L. A. (2000) “A critical role for neutrophil elastase in experimental bullous pemphigoid” J. Clin. Invest. 105:113-123], so a topical application of an elastase inhibitor is a potentially valid treatment for alleviating the skin symptoms of this ailment.
Inhibition of elastase has other effects on the skin that are described in the state of the art. U.S. Pat. No. 7,211,278, for example, describes the use of elastase inhibitors as adjuvants in cosmetic formulations in order to suppress hair growth, for applications such as hair removal.
It is also known that MMPs are involved in perifollicular matrix degradation, and thus, in hair loss. Specifically, cytokines and the epidermal growth factor stimulate MMP-9 production in the lower epithelial compartment of hair root, such mechanism controls capillary follicle involution observed in alopecia [Jarrousse F., Boisnic S., Branchet M. C., Beranger J. Y., Godeau G., Breton L., Bernard B. A. and Mahé Y. F. (2001) “Identification of clustered cells in human hair follicle responsible for MMP-9 gelatinolytic activity: consequences for the regulation of hair growth” Int. J. Dermatol. 40:385-3921 Thus, overexpressed MMP inhibition during alopecic processes could be effective in delaying, and even preventing, hair loss [EP 1 076 549 B1].
Also, MMP activity is related to scar formation in tissues containing collagen. “Scar formation” is defined as the formation of an abnormal morphological collagen structure due to previous injuries or due to the healing process of tissue containing collagen on the skin.
Healing processes consist of three stages: (1) inflammation, (2) tissue formation and (3) tissue remodeling. A necessary stage in the healing process is extracellular matrix degradation: in order for the cells to proliferate in the wounded area and regenerate it, it is necessary that the extracellular matrix be degradated. Such degradation is made through MMPs. Healing process stages are regulated by a balance between the different MMPs and it has been described that an excess of MMP activity causes chronic ulcers. For example, an overexpression of MMP-8 can be associated to the pathogenesis of leg chronic ulcers. Likewise, diabetic ulcers are characterized by a prolonged inflammation, decrease collagen synthesis and high MMP levels.
Scars have different causes (accidents, surgery, skin diseases, burns, acne, infections and accidents in general), but not all scars are the same. Different kinds of scars can be grouped in                Flat and pale scars: formed as a result of the body's natural healing process.        Sunken scars: formed by skin attached to deeper structures, such as muscles, or due to loss of fat in internal tissues. These scars are recessed into the skin and are usually the result of an injury.        Hypertrophic scars: appear when the body produces an excess of collagen during the healing process. These scars elevate over the skin surface and contain irregularly organized collagen.        Keloid scars: formed as a result of an imbalance in the production of collagen during the healing process. These scars not only elevate over skin surface, but also they extend beyond the boundary of the original wound and can continue to grow indefinitely.        Acne scars: formed in skin affected by acne. The scar can be sunken or become a keloid. People who have had chicken pox can have similar scars.        Stretched scars: occur when the skin around a healing wound is put under tension during the healing process. Initially, the scar may appear normal but can widen and thin over a period of weeks or months. This can occur when the wound is close to a joint and is stretched during movement or it may be due to poor healing because of general ill health or malnutrition.        Stretch marks: develop when the skin is stretched rapidly, for example during pregnancy or the adolescent growth spurt.        
Therefore, skin scar reduction is desirable both from the pathological point of view, as healing during fibrotic processes, and from the cosmetic point of view, as in the case of softening the aspect of scars caused by acne or stretch marks. Potential benefits of the inhibition of elastase in relation to scarring have also been described. Thus, there is a degradation of elastin in areas around scars caused by acne vulgaris [Dick G. F., Ashe B. M., Rodgers E. G., Diercks R. C. and Goltz R. W. (1976) “Study of elastolytic activity of Propionibacterium acnes and Staphylococcus epidermis in acne vulgaris and in normal skin” Acta Derm. Venereol. 56:279-282]. Therefore, an elastase inhibitor would be effective as an adjuvant in a formulation of topical application to prevent the appearance of scars in skin affected by acne. Moreover, U.S. Pat. No. 5,922,319 describes the application of an elastase inhibitor to prevent the formation of scars on the cornea in cases of eye trauma.
It has also been described that during adipocytes proliferation and differentiation, MMPs are overexpressed [Traurig M. T., Permana P. A., Nair S., Kobes S., Bogardus C. and Baier L. J. (2006) “Differential expression of matrix metalloproteinase 3 (MMP3) in preadipocytes/stromal vascular cells from nonobese nondiabetic versus obese nondiabetic Pima Indians” Diabetes 55:3160-3165]. Thus, the collagen network of the skin affected by cellulite is destroyed, which is one of the causes of the orange peel skin aspect. Therefore, supplementing the skin with compounds able to stimulate collagen synthesis will help to reduce such collagen destruction and become an effective anti-cellulite treatment. Inhibition of elastase also helps to alleviate the effects of collagen degradation, for example preventing the appearance of marks as a result of aging and improving skin irregularities such as the orange peel skin that is characteristic of cellulite.
MMP activity is also responsible for the extracellular matrix disorganization that surrounds lymphatic and blood vessels. Matrix deterioration around blood vessels allows for a passive vasodilatation which gives place to capillary visibility or telangiectasia, or couperosis. Besides, this microcapillary passive dilatation can cause local blood vessel bursts which can give place to bags under the eyes or dark circles in the periorbital area. Furthermore, MMPs have an influence over vein wall mechanical properties, which can make veins fragile and consequently lead to the development of varicose veins.
Apart from the relation of MMPs to tissue matrix degradation, it has been suggested that MMPs are also involved in different pathologies that concur with an abnormal metabolism of the connective tissue or basal membrane matrix such as arthritis (rheumatoid arthritis, osteoarthritis, etc), bone diseases (osteoporosis, etc.), ectopic angiogenesis, multiple sclerosis, tumors metastasis and tissue ulcers (cornea, stomach, epidermis, etc.) [EP 0 927 161 B1]. Therefore, an MMP inhibitor could be effective in treating and preventing those pathologies caused by an abnormal metabolism of the tissular matrix.
The importance of the presence of collagen and elastin fibers in the skin, mucous membranes and/or scalp and the importance of maintaining, or even reinforcing, their presence can thus be appreciated. It is thus important to have available products whose effects are directed towards maintaining the levels and integrity of collagen and/or elastin in the skin and maintaining the skin's smooth and taut appearance reducing, delaying and/or preventing the signs of aging and/or photoaging. Retaining an elevated elastin and/or collagen content in the skin or increasing the elastin and/or collagen content in the skin may be achieved in various different ways. On one hand, substances which inhibit matrix proteases may be used. On the other hand, however, it is also possible to use substances which increase collagen and/or elastin synthesis in order, by de novo synthesis, to counter the negative effects of such protease-induced collagen and/or elastin degradation. The reduction in de novo protein synthesis which accompanies increasing age may here be at least partially compensated by using active ingredients which increase collagen and/or elastin synthesis.
In the context of this invention the terms “aging” and “aging skin” are used to describe the emergence of visible changes in skin appearance as well as those perceptible by touch, such as for example and in a non-limiting sense wrinkles, fine lines, roughness, expression lines, stretch marks, discontinuities, furrows, flaccidity, skin sagging, such as cheeks sagging, eye pouches, double chin, increase of pore size, loss of elasticity, loss of resilience, loss of firmness, elastosis, anomalous differentiation, hyperkeratinization, keratosis, changes of the skin color, such as marks, reddening or bags under the eyes, appearance of hyperpigmented areas such as age spots, melasma or freckles, loss of smoothness, orange-peel skin, loss of collagen structure and other histological changes of the stratum corneum, of the dermis, epidermis, vascular system (for example the appearance of spider veins or telangiectasias) or of those tissues close to the skin. Skin aging is a process with two main components: chronological, which is due to the passage of time, and photo-induced, which is due to the level of exposure to ultraviolet (UV) radiation and which is known as photoaging. The sum of several environmental factors such as exposure to tobacco smoke, exposure to pollution, and climatic conditions like cold and/or wind also contribute to skin aging.
There are descriptions in the state of the art of various vegetable extracts with elastase inhibitory activity [e.g. JP 11-246386, JP 2000-072649, JP 11-279041, U.S. Pat. No. 6,395,261, U.S. Pat. No. 6,238,674], and synthetic chemical compounds with this activity [U.S. Pat. No. 4,643,991, U.S. Pat. No. 5,008,245, U.S. Pat. No. 5,162,307, U.S. Pat. No. 5,189,178]. Particularly, the U.S. Pat. No. 4,665,053 describes synthetic lipopeptides rich in alanine and proline, in particular lipopeptides containing the L-Ala-L-Ala dipeptide, as elastase inhibitors.
Individual substances which are frequently mentioned in connection with increasing collagen synthesis and are thus prior art are for example active ingredients such as ascorbic acid and the derivatives thereof such as in particular ascorbyl palmitate, magnesium ascorbyl phosphate, sodium ascorbyl phosphate and ascorbyl alpha- and beta-glucoside, retinol and derivatives of retinol such as retinoic acid, retinal, retinol, retinyl acetate or retinyl palmitate or vegetable extracts such as for example extracts of Aloe and Centella species. Active ingredients which are furthermore frequently used to stimulate collagen synthesis also include peptide substances and the derivatives thereof such as e.g. carnitine, carnosine, creatine, matrikine peptides (e.g. lysyl-threonyl-threonyl-lysyl-serine) and further peptide structures such as palmitoylated pentapeptides (e.g. Matrixyl® from Sederma/Croda) or the oligopeptide with the trade name Vincipeptide (from Vincience, France). Moreover, compounds such as asiatic acid, madecassic acid, madecassoside, asiaticoside, extracts of Centella asiatica, niacinamide, astaxanthine, glucans e.g. from yeasts and oats, soy extract and soy isoflavones, such as genistein and daidzein, rutin, chrysin, morin, betel nut alkaloids, forskolin, betulinic acid, extracts of Plantago species, TGF-beta, extracts of Ginkgo biloba, glutamine and glycolic acid are used as collagen synthesis stimulators.
This invention describes synthetic peptides containing uncoded amino acids effective in inhibiting elastase and/or stimulating collagen synthesis in the skin, mucous membranes and/or scalp. No peptide with uncoded amino acids in its sequence presenting elastase inhibitory activity and/or stimulating collagen synthesis in the skin, mucous membranes and/or scalp exists in the state of the art. The use of uncoded amino acids makes their recognition by proteases difficult, increasing the half-life of the peptides that contain them. This increase in the half-life of the peptide extends their efficacy in inhibiting elastase and/or stimulating collagen synthesis.
Hence, despite the large array of existing compounds and/or extracts with an activity against the enzymes that degrade either collagen or elastin or with a potential for estimulating the endogenous synthesis of either collagen or elastin in the skin, mucous membranes and/or scalp, there is still a need to identify new elastase inhibitors and/or collagen synthesis stimulators that are more effective and more selective than those known in the state of the art.